Enumeration of reticulocytes, i.e., the most immature erythrocytes, in human peripheral blood is a valuable component of diagnostic hematology, useful in the diagnoses of hemorrhage, anemia, monitoring bone marrow transplantation, monitoring patients undergoing chemotherapy and other disorders involving blood cell production [U.S. Pat. No. 5,360,739; H. Shapiro, Practical Flow Cytometry, 3.sup.rd edit., 1995; Wiley-Liss, New York; Davis et al, (1990) Pathobiol., 58:99-106; Hoy, (1990) Bailliere's Clin. Haemat., 3:977-988; H. J. Tanke, "Reticulocytes and Mature Erythrocytes" in Flow Cytometry in Haematology (1992) Academic Press Ltd., pp. 75-93]. Because reticulocytes contain ribonucleic acid (RNA), if stained with RNA binding dyes, these cells fluoresce when illuminated by a light source of appropriate wavelength. RNA binding dyes have been used to distinguish reticulocytes from more mature red blood cells (RBCs).
Using fluorescence measurements, distribution of relatively large reticulocyte population can be determined by flow cytometry in a fast and reliable manner provided the cells are appropriately stained with a fluorescent dye. Two of the primary factors that determine the usefulness of a specific reticulocyte enumeration method are: (1) the fluorescence characteristics of the dye when bound to RNA, and (2) method of staining.
A fluorescent dye suitable for reticulocyte enumeration should ideally have bright fluorescence when bound to RNA and very little fluorescence when unbound to RNA. A staining method ideally should be fast, most preferably on the order of one minute or less, if a reticulocyte assay is intended for automated/routine hematology application. Matching the above two requirement has been at the core of research efforts in developing useful reticulocyte assays over the last several years [H. J. Tanke, Reticulocytes and Mature Erythrocytes" in Flow Cytometry in Haematology (1992) Academic Press Ltd.].
Prior art reticulocyte enumeration methods suitable for automated hematology is based on acridine orange or thiozole orange derivatives. Specific examples for Acridine Orange (AO) are cited in Vander et. al., (1993) J. Lab. Clin. Med, 62:132, Thaer et. el. (1970) "Microfluorometric analysis of the reticulocyte population in peripheral blood of mammals" in Cytology Automation, DMD Evans (ed.), E&S Livingstone, Edinburgh (1970), pp. 180-195; and Seligman et. al., (1983) American J. Hematol., 14:57-66], Thiazole Orange (TO) in Cytometry (1986) 7:508 L. G. Lee et. al.; U.S. Pat. Nos. 4,883,867 and 4,957,870; Van Hove et. al. (1990) Clin. Lab. Hemat., 12:287-299; Carter et. al. (1989) Clin. Lab. Haemat, 11:267-271], and coriphosphine-O (CPO) [U.S. Pat. No. 5,639,666]. AO, TO, CPO are all brightly fluorescent when bound to RNA. Each of these dyes are cell membrane permeant, i.e. they transport through the membrane of the reticulocyte cells freely. As a result, these dyes can stain the intra-cellular RNA in relatively short time. This makes them attractive and useful for automated hematology application. However, the AO and CPO suffer from a serious disadvantage in that they tend to stain non-specifically with most things that they contact, including tubing in a flow cytometer. This complicates measurements on cells because fluorescence from the non-specifically bound molecules interfere with RNA bound molecules. As for TO, its use is limited in that it can not be excited in the red wavelengths. This precludes it from being used for fluorescence based cellular analysis using low cost illumination sources such as diode lasers.
There have been research efforts to overcome the limitations presented by the above dyes by developing alternative nucleic acid staining dyes. As a result, there now exists a large number of nucleic acid binding dyes that have fluorescence properties which are least equivalent to those of AO, TO and CPO. Examples of such dyes such as TOTO-1, TOTO-3, YOYO-1, YOYO-3, TO-PRO-1, TO-PRO-3, propidium iodide (PI), ethidium bromide. Unfortunately, none of these dyes are cell membrane permeant. Prior art methods for reticulocyte enumeration using membrane impermeant dye Pyronin Y showed that cells must be fixed for 2 hrs or longer (up to 24 hours). This makes Pyronin Y unsuitable for automated hematology applications [H. J. Tanke, "Reticulocytes and Mature Erythrocytes" in Flow Cytometry in Haematology (1992) Academic Press Ltd., pp. 81]. A second prior art method using cell impermeant dyes such as TO-PRO-3 is described in U.S. Pat. No. 5,563,070. The method requires incubation of a dye in whole blood for at least 30 min. As a result, this method is not suited to automated hematology applications.
Thus, there exist a need in the art for composition and methods that enable rapid staining of intra cellular nucleic acid by normally membrane impermeant fluorescent dyes, and permit ready, accurate and reproducible enumeration of reticulocytes.